1. Field
This invention is concerned generally with the immobilization of enzymes onto water-insoluble carrier materials and specifically with the covalent bonding of enzymes to high surface area, porous inorganic support materials in such a manner that the immobilized enzymes demonstrate retention of enzymatic activity and stability.
2. Prior Art
The desirability of fixing active enzymes on essentially water-insoluble support materials to form readily removable and reusable composites is well recognized. Since enzymes are highly specific in promoting certain chemical reactions, their use, especially in immobilized and hence, reusable form, has very practical appeal in laboratory and industrial applications.
Enzymes have been attached by a variety of methods to numerous water-insoluble support materials, both organic and, more recently, inorganic materials. Methods for immobilizing enzymes on various inorganic materials are described in U.S. Pat. No. 3,556,945 (adsorption on siliceous materials). In U.S. Pat. No. 3,519,538, there are disclosed methods for covalently bonding enzymes to numerous inorganic carriers via intermediate silane coupling agents. Methods of bonding enzymes to porous inorganic carriers having an average pore diameter range which maximizes enzyme loading and half life are described in patent applications Ser. No. 332,807, now U.S. Pat. No. 3,850,751, and Ser. No. 332,804, now U.S. Pat. No. 3,841,971, respectively entitled "Enzymes Immobilized on Porous Inorganic Support Materials" and "Synergistic Enzymes Adsorbed Within Porous Inorganic Carriers". Both of the above applications were filed on Feb. 16, 1973 in the name of R. A. Messing and are assigned to the present assignee. In U.S. patent application Ser. No. 454,140, filed on Mar. 25, 1974, in the name of R. A. Messing, entitled "Method of Immobilizing Urease on Porous Titania", and assigned to the present assignee, methods of pretreating porous titania with stannous ions to maximize loading and half-life of the urease are disclosed.
Although the above disclosures show both the adsorption and chemical coupling of enzymes to inorganic support materials, it can be appreciated that there exist various advantages and disadvantages with each system. For example, as a general rule, adsorbed enzyme systems are relatively inexpensive to prepare and use. On the other hand, adsorbed enzymes are fixed to the carrier by relatively weak bonds and, hence, can detach relatively easy. One method for minimizing such detachment, as shown in Ser. No. 332,807, involves using porous inorganic carriers having an average pore diameter which is very small, e.g., less than 1000 A, very preferably, less than about 500 A.
In the case of enzymes coupled chemically to inorganic carriers via covalent bonds (e.g. U.S. Pat. No. 3,519,538, disclosing the use of an intermediate silane coupling agent), the enzymes are fixed to the surface relatively firmly. On the other hand, such chemically coupled enzyme systems are relatively time consuming and expensive to prepare and use.
I have now prepared an immobilized enzyme system using inorganic carriers which has many of the advantages of past systems with minimal disadvantages in that the enzymes are chemically coupled to the inorganic carrier but in a relatively simple and economical manner. The immobilized enzyme composites and methods for preparing them are described in detail below.